PROJECT SUMMARY Nearly all Gram-positive bacteria synthesize a transmembrane, Ser/Thr kinase containing 3 to 5 extracellular PASTA-domains (i.e. a ?PASTA kinase?) that controls critical processes including antibiotic resistance, toxin production, virulence, or cell division; in some bacteria the PASTA kinase is essential for viability. As such, PASTA kinases represent attractive targets for new therapeutics. However, a basic understanding of the mechanisms by which kinases in this family function to perceive environmental stimuli in vivo and process that information to coordinate adaptive biological responses is lacking. Such information is critical to inform development of new therapeutic approaches. The research proposed here seeks to help address this gap by elucidating fundamental aspects of function for a representative kinase in this family, the IreK kinase in Enterococcus faecalis, which we have shown is required for intrinsic resistance of E. faecalis to cell-wall-active antimicrobials and to detergents present in bile, such as cholate. This research uses genetic and biochemical approaches coupled with state-of-the-art mass spectrometry strategies to overcome key roadblocks to progress by defining the extent and functional impact of phosphorylation on IreK in vivo, and by identifying downstream substrates for phosphorylation by IreK. Completion of these studies will enable us to take important steps forward in understanding how IreK functions in E. faecalis cells. Given the conserved domain architecture among the family of PASTA kinases, it is likely that insights from this work will translate to other PASTA kinases as well.